Neurons and endocrine cells secrete transmitter or hormones in Ca- triggered bursts of exocytosis. In a broad sense, exocytosis is how neurons and neuroendocrine cells "talk". Secretion involves four steps: (i) Making the secretory vesicle and filling it with cargo, (ii) transporting the vesicle to a site of exocytosis, (iii) membrane fusion or exocytosis, and finally (iv) the retrieval of the vesicle's membrane by mechanisms broadly called endocytosis. In synaptic vesicles, a new synaptic vesicle is made from the membrane thus retrieved, and the four events form a cycle. All four steps involve multiple proteins and multiple layers of regulation. All four can fail in disease, and most or all neurologic diseases ultimately result in the failure of one or more steps in the synaptic vesicle cycle. This grant proposes functional studies of exo- and endocytosis at the level of single secretory vesicles in intact neurons and endocrine cells. Using an unconventional light microscopy we helped to adapt for this purpose, we will specifically and simultaneously mark proteins in exo- and endocytic vesicles in different fluorescent colors, and then observe the recruitment and release of these proteins to vesicles, and the recruitment and release of vesicles from the plasma membrane. Through observations in living cells, we focus on the following questions. (1) How are synaptic vesicles transported and recruited to their docking sites at presynaptic terminals? (2) How completely do secretory vesicles fuse with the plasma membrane? (3) How do neurons and endocrine cells deal with the material that exocytosis has inserted into the plasma membrane, and is such material inserted in the first place? The planned experiments will generate direct evidence in a way not possible with other known methods, and will address fundamental questions in cellular neurobiology.